This R15 study will use quantitative LC?MS/MS assays for cytoplasmic peptidoglycan (PG) intermediates to 1) study vancomycin (Vm) resistance in Enterococcus spp. (VRE), and 2) as an aid to the identification of novel cell wall biosynthesis targeted anti?VRE agents from chemical library screening. This study will be driven by two key innovations. The first is our development of quantitative LC?MS/MS?based methods for the peptidoglycan intermediates in both the normal and alternative PG biosynthesis pathways in VRE. Preliminary studies in VRE reveal that PG metabolite levels change rapidly in response to Vm exposure, whereas mRNA levels measured by RT?qPCR respond much more slowly. The second innovation is the development of a novel approach to increase library diversity by in situ metabolism of libraries using human liver microsomes. Screening of the original library identifies agents active in their un?metabolized form, and screening of the ?pre?metabolized? library identifies compounds with active metabolites ? which are then identified. Such a strategy is of particular interest when applied to an FDA approved drug library, as proposed in this study. It is proposed in AIM 1 to characterize the effect of Vm on PG metabolite and VanA/B mRNA levels in a collection of VanA? and VanB?type resistant Enterococcus strains. This will determine the range of PG pathway behavior among this group of organisms, and clarify the relative contributions of metabolite level changes vs gene expression changes to resistance. This aim will also explore the effect of a number of other cell wall biosynthesis targeted agents on metabolite and mRNA levels, which will provide additional insight into PG biosynthesis in VRE and reference data for new inhibitor characterization. In AIM 2 of this study, we will perform whole cell based chemical library screening to identify novel anti?enterococcal agents. Two libraries, including an FDA approved drug library, will be used for this effort. Encouraging preliminary screening data is presented. The un?met/pre?met screening approach applied to the FDA approved drug library has the potential to identify FDA approved drugs, or metabolites of FDA approved drugs, as novel new anti? enterococcal agents. In AIM 3 active agents identified in this screen will be tested to identify those acting on the PG biosynthesis pathway, and the best PG pathway and non?PG pathway inhibitors further characterized for mechanism of action.